Int. J. Morphol., 30(3):1007-1012, 2012. Metallothionein: An Overview on its Metal Homeostatic Regulation in Mammals Metalotioneina: Una Visión General de su Regulación Homeostática de Metales en Mamíferos Natthiya Sakulsak SAKULSAK, N. Metallothionein: an overview on its metal homeostatic regulation in mammals. Int. J. Morphol., 30(3):1007-1012, 2012. SUMMARY: Metallothionein (MT) is a ubiquitous protein with a low molecular weight of 6-7 kDa weight and it was first identified in the kidney cortex of equines as a cadmium (Cd)-binding protein responsible for the natural accumulation of Cd in the tissue. The mammalian MT contains 61 to 68 amino acid residues, in which 18 to 23 cysteine residues are present. The expression of MT starts by binding of metal transcription factor-1 (MTF-1) to the regulative region of MT gene called metal responsive elements (MREs). The induction of MT through the MREs region can be initiated by several metal ions such as zinc (Zn), copper (Cu) and Cd. However, Zn is the only heavy metal which can reversibly and directly activate the DNA-binding activity of MTF-1. In mammals four types of MT are expressed and they are termed metallothionein-1 (MT1), metallothionein-2 (MT2), metallothionein-3 (MT3), and metallothionein-4 (MT4). MT1 and MT2 are expressed in almost all tissues while MT3 and MT4 are tissue-specific. MT is a key compound involved in the intracellular handling of a variety of essential and nonessential post-transitional metal ions. In order to the heavy metal binding ability of MT, it is suggested to play roles both in the intracellular fixation of essential trace elements Zn and Cu, in controlling the concentrations, and in neutralizing the harmful influences of exposure to toxic elements. KEY WORDS: Metallothionein; Cadmium; Zinc. INTRODUCTION Metal-binding proteins have been found in various several biological and chemical signals. Therefore, the mammalian and non-mammalian tissues. The first mammalian potential role of MT in health and disease is evident. MT was discovered by Margoshes & Vallee (1957). It was However, a huge number of factors stimulating the isolated from the horse kidney cortex of equines and revealed biosynthesis of MT make it difficult to identify its specific to have a high affinity to Cd. This protein was later biological role. In fact, the expression of MT gene in higher biochemically characterized and termed “metallothionein” species is stimulated by a variety of factors including heavy (MT), due to its high content of metals and cysteine residues metals, glucocorticoids, hormones, oxidants, strenuous (Kagi & Vallee, 1960; 1961). Its three-dimensional protein exercise and cold exposure. The induction of MT by heavy structure has been reported by both X-ray crystallography metals and its subsequent metal accumulation in the cell have (Robbins & Stout, 1991) and NMR spectroscopy in the 1990s been used as a biomarker in the field of environmental (Otvos & Armitage, 1991). MT belongs to a superfamily of toxicology (Sakulsak et al., 2009). The expression of MT in intracellular metal-binding proteins which is composed of 15 cells is also induced by superoxide and hydroxyl radicals families, and has various consensuses sequences inferred from generated by g-radiation. It is suggested that MT acts either both amino acid and polynucleotide sequences. MT has been as a scavenger of radicals or a Zn donor for enzymes identified in all animal phyla examined to date and also in participating in the repair processes (Ryvolova et al., 2011). certain fungi, plants and cyanobacteria (Webb & Cain, 1982; Robinson et al., 1993; Coyle et al., 2002) as well as in humans Hence, the precise functional significance of MT is (Miles et al., 2000). still under debates. Some hypotheses have been advanced for the functional significance of mammalian MT, such as In mammalian cells, MT has been known to be the homeostasis and transport of physiologically essential involved in the regulation of Zn- and Cu-level as well as metals (Zn, Cu), the metal detoxification (Cd, Hg), the Department of Anatomy, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand. Centre of excellence on environmental research and innovation, Naresuan university, Phitsanulok, Thailand. Centre of excellence in medical biotechnology, Naresuan university, Phitsanulok, Thailand. 1007 SAKULSAK, N. Metallothionein: an overview on its metal homeostatic regulation in mammals. Int. J. Morphol., 30(3):1007-1012, 2012. protection against oxidative stress, the maintenance of cysteines with three bridging sulfur atoms (Coyle et al., 2002; intracellular redox balances, the regulation of cell Cherian et al., 2003). The MT binding to metals starts with proliferation and apoptosis, the protection against neuronal the a-domain and follows by the b-domain. These injury and degeneration, and the regulation of neuronal arrangements (28 intramolecular Zn-sulfur bonds) account outgrowth (Palmiter, 1998, Miles et al., 2000). These for the very tight Zn- binding in both clusters and for the historical reports about this unique molecule have captured Zn-donating properties of MT. Although the exchangeability the curiosity of biologists and chemists and inferred the depends on metal species, in vivo mammalian MT appears mystery of this MT family for over five decades, as described as the predominant form of ZnMT complexes. (Orlowski & in several reviews (Vasak & Hasler, 2000; Coyle et al., 2002; Piotrowski, 1998). The tertiary structure of MT is dynamic, Cherian et al., 2003; Cherian & Kang, 2006, Thirumoorthy especially for Zn and Cd. The exchange of Zn and Cd occurs et al., 2007; Carpene et al., 2007). rapidly within the b-domain, more slowly in the a-domain and perhaps also exchange with other ions bound to Metal-binding properties of MT. Mammalian MT is a sin- intracellular ligands. Furthermore, MT also donates metal gle chain polypeptide containing 61-68 amino acid residues. ions to ligands with a higher affinity on the other proteins It is a non-enzymatic intracellular protein with a low (Coyle et al., 2002). molecular weight of 6 to 7 kDa (< 7,000 Da) and it is ubiquitous in eukaryotes. The cysteine-containing residues However, it has recently been reported in mammals (18-23 cysteine-residues) comprise about 30% of the that under the metal-excess condition, the N-terminal domain molecule and they juxtapose to basic amino acids such as is responsible for the formation of non-oxidative metal-brid- lysine and arginine without aromatic amino acids or histidine ge dimers. In contrast, under the aerobic condition, a specific residuals. MT binds metals through the thiol (-SH) group of intermolecular disulfide is formed between two C-terminal their cysteine residues. Typically, MT has a high affinity to domains. Both forms of the dimers show radical differences both divalent essential metals such as Zn and Cu, and in the reactive properties of their respective clusters bound nonessential (or toxic) metals such as Cd and Hg which give to the metal ions (Carpene et al., 2007). rise to metal-thiolate clusters (Kagi & Vallee, 1960; Robbins et al., 1991). The MT binding affinity to metal ions is The rate of MT degradation varies among animal different between various metals or metal-dependent as species. The rate of MT degradation is determined by identity reported in several in vitro studies using MT common to rat of metal-atoms bound to the protein, and the half-lives for liver, and it showed a slightly different relative order of the Cd-, Zn- and Cu-MT in the liver have been reported to be affinity to metals as shown in Table I. approximately 80, 20 and 17 hrs, respectively (Richard, 1989). In addition, the differences in the metal distribution Table I. The binding affinity of MT for metal ions. between MT isoforms may also influence the rate of MT degradation. According to its metal binding properties, MT The MT binding affinity for metal ions References has been suggested to be involved in the cellular homeostatic Cd > Pb > Cu > Hg > Zn > Ag > Ni > Co Waalkes et al., 1984 Hg > Cu > Cd > Zn > Ni = Co Nelson et al., 1985 control and the regulation of trace elements (Tapiero & Tew, Hg > Ag >> Cu > Cd > Zn Hamer et al., 1986 2003). GENE REGULATION OF MT. The induction of MT is Moreover, MT have the greatest stability constant specifically metal-dependent. The presence of metal response estimated for Cu (1019-1017) followed by that for Cd (1017- elements (MREs) in the upstream sequences of MT gene 1015) and then Zn (1014-1011), indicating that CdMT has a represents the evidence for this specific metal-induced 100-fold higher stability than ZnMT. According to both transcription of MT (Stuart et al., 1985). The candidate MRE- binding affinity and stability of MT indicate that the other binding protein termed metal transcription factor-1 (MTF- metal ions such as Cd, Hg or Cu are more capable to displace 1), acts as the positive mediator to initiate the expression of Zn to further forming the more stable CdMT, HgMT or MT gene. This factor, MTF-1 required increased Zn CuMT complexes than other low affinity and stability metal concentrations for optimal DNA binding (Westin & ions (Sabolic et al., 2010). Schaffner, 1988). Based on studies of mice (Heuchel et al., 1994; Gunes et al., 1998), MTF-1 is a ubiquitous expressed MT is composed of two distinct clusters: a more stable Zn finger protein that is necessary for basal and heavy me- a-domain closer to C-terminal which incorporates four tal-induced expression of MT. Subsequently, a constitutively divalent metal atoms bound to 11 cysteines with five bridging active MTF-1 is initially inhibited by a Zn-sensitive inhibitor sulfur atoms, and a more reactive b-domain closer to N-ter- termed metal transcription inhibitor (MTI). In the presence minal which contains only three metal atoms bound to nine of Zn, Zn-ions are bound to Zn-finger binding sites, resulting 1008 SAKULSAK, N.